Project Summary Bacillithiol is an antioxidant compound produced by many Gram-positive bacteria including the pathogenic organisms Bacillus anthracis and Staphylococcus aureus. It is involved in the maintenance of redox homeostasis and detoxification of xenobiotic compounds, including the FDA-approved antibiotic fosfomycin. Three enzymes are responsible for the biosynthesis of bacillithiol: BshA (a glycosyltransferase), BshB (a deacetylase), and BshC (a putative cysteine ligase). Although the activities of BshA and BshB have been confirmed in vivo and in vitro, BshC activity has only been inferred in vivo, suggesting that a key component involved in cysteine ligation is missing. Structural analysis of these enzymes is only in its initial stages. The proposed research seeks to use X-ray crystallography to further characterize BshC and to determine substrate, product, or analog-bound structures of all three bacillithiol biosynthesis enzymes. These structures will demonstrate how the enzymes accommodate their substrates in their active sites and give insight into the details of their mechanisms. Functional studies will be conducted on BshA to characterize the manner in which bacillithiol acts as a feedback inhibitor. Methods to produce functional BshC enzyme will be employed, including the utilization of a Gram-positive protein expression system. Additionally, a fluorescence-based assay will be used to characterize a second ligand binding site found within the BshC structure. The knowledge gained from the proposed research will provide a foundational understanding of these enzymes and ultimately give direction to the design and characterization of inhibitors capable of disrupting bacillithiol production in an effort to combat microbial resistance to fosfomycin.